Laboratory Practices - GRE

Sodium fluoride is a serine/threonine phosphatase inhibitor. This is important to have in a cell lysis buffer if the protein of interest is phosphorylated. The presence of sodium fluoride will prevent the phosphoryl group from being removed by a serine/threonine phosphatase before it can be studied. It is often added to the cell lysis buffer along with sodium vanadate, which is a tyrosine/alkaline phosphatase inhibitor.

The correct answer is electrophoretic mobility shift assay (EMSA). In an EMSA, specific DNA sequences are radioactively labelled and incubated with either protein lysates or purified proteins. The protein-DNA mixture is then loaded onto a non-denaturing gel to separate the proteins and DNA by size. DNA that is bound by protein will migrate more slowly than unbound DNA, which will migrate quickly. To detect radioactivity, a film is exposed to the gel, developed and interpreted. It is important to remember that the radioactivity is emitted by the DNA and not the protein.

The correct answer is Western blot. In a Western blot, cell protein lysates are run on a denaturing gel and probed with a specific antibody. The student researchers can detect the existence of the DNA polymerase protein in the cell lysate using a specific antibody to that polymerase.

EMSAs determine a protein's ability to bind a DNA sequence, while electroporation, PCR, and restriction digests are not methods to detect protein expression.

The correct answer is DNA microarray. In a DNA microarray, short DNA sequences (probes) are labelled on a microarray chip. Probes for the same gene are clustered in the same area on the array. cDNA from a sample is labelled with a fluorescent dye and washed over the chip. If a certain gene is more highly expressed, its cDNA will bind the respective probes and give a higher intensity than a lower expressed gene.

The correct answer is whether the C-terminus of the protein is extracellular or intracellular. This is a very common laboratory technique to determine which terminus of a transmembrane protein is intracellular and extracellular. Since the antibody is specific to the C-terminus of the protein and if the C-terminus of the protein is intracellularly localized, we will only be able to detect the protein if the cells are permeabilized. In cells that are not permeabilized, the antibody will not be able to enter the cells, but permeabilization will allow the antibody to pass through the plasma membrane and bind the C-terminus. If the C-terminus is extracellular, the antibody will bind regardless of permeabilization.

All of the given answers are valid methods to introduce exogenous DNA into a cell. When working with bacteria, transformations are a simple way to introduce plasmid DNA into the cell by chemical competency. Electroporation electrocutes both bacterial and cell culture cells to introduce exogenous DNA. Infection of target cells may also be a viable method if the laboratory is equipped to generate live virus containing exogenous DNA to be introduced. Finally, transfection refers to a process mainly reserved for cell culture that introduces exogenous DNA through liposome fusion (containing DNA) with cell membranes.

Microarrays allow for the study of expression levels of thousands of genes simultaneously. Although microarrays do not tell you anything about the actual sequence of the genes, they are the only option for studying the expression levels of massive numbers of genes. It is possible to study expression levels using PCR, but not on a large scale.

Running allozyme gels was one of the first methods used in molecular studies of these enzymes that have slightly different functions due to having slightly different genetic codes (alleles). This term refers specifically to enzymatic variation created by genetic variation/alleles.

The correct answer is to separate proteins and other impurities from DNA or RNA. An extraction by phenol followed by chloroform will separate proteins and other impurities from DNA and RNA (in suspended fraction). Next, depending on the type and concentration of salt used, a researcher can selectively precipitate either DNA or RNA from the mixture with the addition of ethanol. This is a simple method to obtain pure clean ribonucleic acids from impure sources.

The correct answer is lysis of the plasma membrane. While detergents such as sodium dodecyl sulfate are commonly used in western blots as a protein denaturing agent, it also breaks down the plasma membrane by emulsifying membrane-bound lipids and proteins.

The correct answer is introduce membrane pores. Escherichia coli grows optimally at 37 degrees Celcius. However, an increase in temperature causes the plasma membrane to become more fluid, introducing small pores through which plasmid DNA can enter into the cell.

The correct answer is the ampicillin the nutrient agar plates expired. The plasmid expresses beta-lactamase, an enzyme that degrades ampicillin. Successfully transformed bacteria will be able to grow in the presence of ampicillin, but untransformed bacteria should not grow. Since the bacteria in our control transformation do not have ampicillin resistance conferred by beta-lactamase, we would expect that no growth would be observed on any nutrient agar plates with ampicillin. However, we observe uncontrolled growth on these plates, indicating that the ampicillin has expired and is no longer a viable selectable marker for transformed bacteria.

The annealing temperature is the optimal temperature at which the primers bind the template DNA sequence. This temperature takes into account several factors: the number of basepairs in the primers, the relative guanine-cytosine content, and their melting point.

None of the answer choices are correct. In order to successfully clone a bacterial gene into a mammalian expression vector, the following must be completed: First, the bacterial gene should be PCR amplified from bacterial cDNA, which contains only exons. Second, the vector backbone should be digested with restriction enzymes to linearize the vector so that the bacterial gene can be inserted at a specifc locus (multiple cloning site). Third, the amplified bacterial gene and the digested vector backbone are ligated together to create the desired expression construct.

The correct answer is cDNA. Given that this gene most likely has several introns, complementary DNA (cDNA) should be used as a template since it only contains exons. When subcloning a gene into an expression vector, only coding sequence should be present because the transcribed RNA will not be processed like it is in host cells.

Plasmids are circular pieces of DNA that are resistant to exonucleic degradation (exonucleases cut from DNA ends). Bacteria harbor native plasmids that replicate independently from their genome and express genes that often confer a survival advantage. Scientists often clone genes into plasmids and express them in a variety of host cells.

Frederick Sanger invented the chain termination method of DNA sequencing in 1977, which has been widely used since its invention for short DNA sequences. The method is know as Sanger Sequencing. Leroy Hood was responsible for automating this method, Kary Mullis was the inventor of the polymerase chain reaction to create copies of DNA, E. M. Southern lent his name to another DNA study method called Southerns, and Craig Venter was responsible for other later DNA sequencing methods.

Polymerase chain reaction requires two, not one, synthesized primer molecules to correctly amplify a DNA molecule into new copies. One primer attaches to each end of your desired DNA segment, and amplification occurs from these sites by polymerase molecules. The other options are all correct facts about PCR.

Cosmids could handle DNA insert sizes of 25-50kb. BAC's could handle insert sizes of 100-300kb, and YAC's even more. These are still used for tricky genome projects due to this large insert size, like antifreeze protein genes in Arctic fish. Plasmids can generally only handle insert sizes of 5-10kb.

These were all important advances in improvement of the Sanger technique that increased DNA sequence output. Modification of polymerases allowed quicker thermal cycling reactions and incorporation of manufactured bases with fluorescent tags. Four different fluorescent tags for each base allowed for easy identification of different bases. Lastly, capillary chromatography allowed for huge parallelization of DNA sequencing.